The serine protease enterokinase (in short enterokinase or EK), also known as enteropeptidase, is a heterodimeric glycoprotein, a mammalian enzyme catalyzing the conversion of trypsinogen into active trypsin. Enterokinase has preference for the substrate sequence Asp-Asp-Asp-Asp-Lys ((Asp)4-Lys, DDDDK), where it selectively cleaves after lysine. Enterokinase isolated from bovine duodenal mucosa exhibits a molecular weight (MW) of 150,000 and a carbohydrate content of 35 percent. The enzyme is comprised of a heavy chain (MW˜115,000) and a disulfide-linked light chain (MW˜35,000) (Liepnieks et al., J. Biol. Chem., 254(5): 1677-1683 (1979)). The function of the heavy chain is to anchor the enzyme to the mucosal membrane. The light chain acts as the catalytic subunit.
In E. coli many mammalian proteins are expressed as fusion proteins, which have to be cleaved to release the mature, active protein. For that purpose a processing enzyme is needed, preferably one which cleaves directly at the junction leaving no extra amino acids on the product. Enterokinase is such an enzyme, and much effort has been made to establish a recombinant process to obtain enterokinase or enterokinase analogues in E. coli. However, the results so far have been rather poor: Available commercial products are expensive and of low specific activity, due to inefficient renaturation of precipitated EK or inefficient secretion of soluble EK.
A process in E. coli aiming at a soluble EK product leads to a mixture of soluble and insoluble protein, requiring 2 routes of purification, expensive affinity columns and low yields altogether. In order to get a uniform product, the EK has to be produced as insoluble material in inclusion bodies. They are easy to isolate but challenging to renature in satisfactory yields, due to possible aggregation of the protein.
An object of the invention is to obtain a mammalian enterokinase analogue with improved properties.